Field of the Invention
The present invention relates to thermostability of a cellobiohydrolase enzyme. A cellobiohydrolase is one of glycoside hydrolyzing enzymes involved in a process of hydrolyzing lignocellulose such as cellulose and hemicellulose and generating monosaccharides. More specifically, the present invention relates to a novel thermostable cellobiohydrolase, a polynucleotide encoding the aforementioned thermostable cellobiohydrolase, an expression vector for expressing the aforementioned thermostable cellobiohydrolase, a transformant into which the aforementioned expression vector has been incorporated and a method for producing a cellulose degradation product using the aforementioned thermostable cellobiohydrolase.
Priority is claimed on Japanese Patent Application No. 2014-050084, filed Mar. 13, 2014, the content of which is incorporated herein by reference.
Description of the Related Art
Plant biomass or lignocellulose is the most abundant renewable energy source on the earth and is expected as an alternative resource to petroleum. Main components of the biomass based on the dry weight are polysaccharides, such as cellulose and hemicellulose, and lignin. For example, polysaccharides are hydrolyzed to monosaccharides, such as glucose and xylose, by glycoside hydrolases collectively called as cellulase enzymes, and are then used as biofuels or materials for chemical products.
Lignocellulose having a complex structure is persistent and is difficult to degrade or hydrolyze with a single enzyme. For the complete degradation of lignocellulose, in general, three types of enzymes, i.e., an endoglucanase of glucoside hydrolase (cellulase or endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase. EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21) are believed to be required. For the hydrolysis of lignocellulose, it is considered that appropriate formulation of multiple enzymes is necessary, including, in addition to the above, a xylanase serving as a hemicellulase (endo-1,4-β-xylanase, EC 3.2.1.8) or other plant cell wall degrading enzymes. On the other hand, it is thought that it is possible to significantly reduce the enzyme costs by using a thermostable enzyme and performing a lignocellulose hydrolysis process at a high temperature, thereby considerably reducing the enzyme amount and hydrolysis time. For this reason, for various cellulases, development of enzymes that are more excellent in terms of thermostability has been desired.
For thermophilic filamentous fungi that are eukaryotes, as compared with thermophilic bacteria and hyperthermophilic archaea that are prokaryotes, their threshold temperature for survival is as low as about 55° C. Therefore, in general, the thermostability of glycoside hydrolases expressed and secreted by thermophilic filamentous fungi is not so high. Cellobiohydrolases CBHI and CBHII of a thermophilic filamentous fungus Chaetomium thermophilum exhibit optimum temperatures of 750° and 70° C., respectively (for example, see Non-Patent Document 1), and a cellobiohydrolase CBHI of Thermoascus aurantiacus exhibits an optimum temperature of 65° C. (for example, see Non-Patent Document 2), which are the highest thermostability that has been reported so far for the cellobiohydrolases derived from filamentous fungi. Although there is a method of further improving the thermostability by substituting one or more amino acids in the cellobiohydrolase (for example, see Patent Document 1 or 2), the thermostability of the thus obtained mutant cellobiohydrolase is still at an insufficient level.
On the other hand, thermophiles growing at or above 55° C. and hyperthermophiles growing at or above 80° C. have been isolated and cultured from the extreme environments such as hot springs, hydrothermal vents, oil fields and mines. The majority of thermostable glycoside hydrolases derived from these thermophilic bacteria and hyperthermophilic archaea are enzymes with an endoglucanase activity, xylanase activity, xylosidase activity or glucosidase activity. Only a few cellobiohydrolases that play the most important role in the lignocellulose hydrolysis process have been isolated from three kinds of thermophilic bacteria belonging to the genera Clostridium, Thermobifida and Thermotoga. For example, a thermophilic anaerobic bacterium Clostridium thermocellum presents an enzyme complex cellulosome with a high lignocellulose hydrolytic activity extracellularly. The main enzymes of a cellulosome are cellobiohydrolases, and the three types thereof consisted of CelO belonging to GH5 family and CbhA and CelK belonging to GH9 family have been isolated, all of which have an optimum temperature (Topt) of 60 to 65° C. (for example, see Non-Patent Documents 3 to 5). Two types of cellobiohydrolase genes; i.e., E3 belonging to GH6 family (for example, see Non-Patent Document 6) and Cel48A belonging to GH48 family (for example, see Non-Patent Document 7) have been isolated from a thermophilic actinomycete Thermobifida fusca. These cellobiohydrolases exhibit relatively high thermostability, exhibit a 50% activity of the maximum value within a temperature range from 40 to 60° C. and exhibit stable activity at 550° for at least 16 hours. However, these two types of cellobiohydrolases exhibit insufficient activity at 70° C. or higher, thus in the case of carrying out a hydrolysis process of cellulose by using these, the upper limit for the temperature of hydrolysis process will be from 60 to 65° C. It has been reported that a cellobiohydrolase derived from a thermophilic bacterium belonging to the genus Thermotoga exhibited the highest thermostability, with an optimum temperature (Topt) of 105° C. and a half life of activity (Thalf) of 70 minutes at 108° C. (for example, see Non-Patent Document 8). However, the above enzyme exhibits endoglucanase-like substrate specificity and exhibits a degradation activity only against amorphous cellulose and carboxymethyl cellulose (CMC). Further, since the hydrolytic activity of the filter paper is weak, efficient hydrolysis of crystalline lignocellulose by the above enzyme cannot be expected.